1. Field of the Invention
The present invention is related to a method to produce poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) by the recombinant Escherichia coli, in particular, overexpression of phaCAB-vgb-prpE gene by the recombinant Escherichia coli to produce PHBV.
2. Description of the Prior Art
Polyhydroxyalkanoates (PHAs) are high molecular weight polymers that can be used as biopolymers, and are biocompatible, absorbable, and biodegradable. PHAs can also be used as biomedical materials, e.g. implants, films or suturing materials. The monomers of PHAs, HA, are linked by ester bonds. A methyl or ethyl group in its C3 position will result in the formation of polyhydroxybutyrate (PHB) or polyhydroxyvalerate (PHV), respectively, whereas polymerization both of PHB and PHV gives rise to poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) with the structural form

PHBV can be used as biodegradable plastics. Due to its extraordinary biocompatibility and thermoplasticity, at present, PHBV is broadly used to produce various types of PHBV films for medical or pharmaceutical use, drug capsules, bone filler, or surgical stitches.
It was known that Ralstonia eutropha can import large amounts of propionate into cells by using propionate permease (PrpP) and can synthesize propionyl CoA from propionate by using propionyl-CoA synthetase (PrpE); hence, the addition of propionate in the bacteria culture medium can induce the formation of 3-ketovaleryl-CoA by facilitating the reaction between propionyl-CoA and acetyl-CoA using preexisted Beta-ketothiolase B (BktB), which consequently resulted in hydroxyvalerate (HV) after the process of reduction catalyzing by the reductase, and said HV can react with hydroxybutyrate (HB) to form PHBV.
Various properties of PHBV including tensile strength, elastic modulus, and crystallinity will decrease accordingly with increased HV amount, while its elasticity and flexibility will notably improved. However, the amount of PHBV synthesized by bacteria in nature is usually very low (less than 5%). In the case of using wild strains in culture medium with propionate to produce PHBV, the growth of most microorganisms will be inhibited which consequently restricts the yield and the availability of the product. Previous studies have demonstrated co-transformed of E. coli with plasmids which one carries phaCAB gene (for PHB synthesis) and the other carries bktB, prpP or prpE gene (relevant genes involved in the metabolism of propionate); yet, culturing of the resulted recombinant E. coli requires two different antibiotics in the growth medium which results in the cost rise and the cost is a major concern in practical production. Furthermore, the numbers and ratios of the two plasmids in E. coli cannot be controlled, and the amount of HV produced by the recombinant E. coli was only 15˜20% (mol %) of total PHBV.
The transformed recombinant E. coli containing only one plasmid carries both phaCAB and prpE genes disclosed in present invention. Also, the amount of HV in total PHBV produced by the recombinant E. coli strain disclosed in the present invention is regulated by the concentration of propionate in the culture medium, and is around 35%. Thus, the method to produce PHBV by microorganisms disclosed in the present invention has clearly demonstrated the significant improvements.